Vasoactive intestinal peptide (VIP) is a 28 amino acid polypeptide with a broad spectrum of biological functions. The effects of VIP are mediated by high affinity receptors. cDNA cloning of the VIP receptor showed that it is a G-protein coupled receptor containing seven transmembrane domains, and that it is expressed in a variety of tissues. The long term objective of this proposal is to understand the mechanisms that regulates VIP receptor gene expression during development and in disease. The Specific Aims in the original KO8 Award (DK02346) were: (1) To isolate and characterize the rat VIP receptor gene. (2) To identify cis-acting elements and DNA binding proteins important for transcriptional regulation of the VIP receptor gene in lung cells. (3) To study regulation of VIP receptor expression during rat development. (4) To study regulation of VIP receptor expression in carcinoma cell lines. The goals for the current proposal are to carry out those studies in the specific aims list above that have not been accomplished in the first three years of the KO8 Award. The Specific Aims are: (1) To map the DNA binding and trans-repession domains of VIPR-RP, and determined the functional importance of the phosphorylation sites. To map the DNA binding domain of VIPR-RP, different regions of the protein will be expressed in and purified from E.coli. The recombinant protein will be use in gel mobility shift assays with the VIPR-RP binding site as the probe. The trans-repression domain will be determined by making fusion constructs containing various regions of the VIPR-RP and the Gal- 4 DNA binding domain. The transcription repression activity of each construct will be tested by co-transfecting Cos7 cells with a reporter plasmid containing binding sites for Gal4 and luciferase. The functional importance of each putative sites for protein kinases within the DNA binding or trans-repression domain will be determined by in vitro kinase assays, and mutagenesis. (2) To generate DNA constructs that will be used for targeted disruption of the VIPR1 gene. A mouse genomic library will be screened using the rat VIPR1 gene as a probe to isolate the mouse VIPR1 gene. A Cre-loxP recombination approach will be used. A targeting vector containing two lox sites, and a vector containing a cell type-specifically expressed Cre transgene will be generated. These DNA constructs will be used in the future to generate transgenic mice that contain either null mutation or cell type-specific disruption of the VIPR1 gene. These studies will provide important insight into the physiological functions of VIP and the molecular mechanisms that regulates VIP receptor expression.